Peptide-based gemini compounds

ABSTRACT

New peptide-based gemini compounds comprising two linked chains:  
                 
 
     each chain having:  
     (1) a positively charged hydrophilic head, Q 1  or Q 2 , formed from one or more amino acids and/or amines  
     (2) a central portion, P 1  or P 2 , having a polypeptide backbone, and  
     (3) a hydrophobic tail, R 1  or R 2 ,  
     the central sections of each chain being linked together by bridge Y through residues in P 1  and P 2 ,  
     are disclosed.  
     Methods for their preparation and uses are also disclosed. Such uses include transfection of polynucleotides into cells in-vivo and in-vitro.

[0001] This invention relates to newly identified peptide-based geminisurfactant compounds, to the use of such compounds and to theirproduction. The invention also relates to the use of the peptide-basedgemini compounds to facilitate the transfer of compounds into cells fordrug delivery.

[0002] Surfactants are substances that markedly affect the surfaceproperties of a liquid, even at low concentrations. For examplesurfactants will significantly reduce surface tension when dissolved inwater or aqueous solutions and will reduce interfacial tension betweentwo liquids or a liquid and a solid. This property of surfactantmolecules has been widely exploited in industry, particularly in thedetergent and oil industries. In the 1970s a new class of surfactantmolecule was reported, characterised by two hydrophobic chains withpolar heads which are linked by a hydrophobic bridge (Deinega, Y et al.,Kolloidn. Zh. 36, 649, 1974). These molecules, which have been termed“gemini” (Menger, F M and Littau, C A, J.Am. Chem. Soc. 113, 1451,1991), have very desirable properties over their monomeric equivalents.For example they are highly effective in reducing interfacial tensionbetween oil and water based liquids and have a very low critical micelleconcentration.

[0003] Cationic surfactants have been used inter alia for thetransfection of polynucleotides into cells in culture, and there areexamples of such agents available commercially to scientists involved ingenetic technologies (for example the reagent Tfx™-50 for thetransfection of eukaryotic cells available from Promega Corp. WI, USA).

[0004] The efficient delivery of DNA to cells in vivo, either for genetherapy or for antisense therapy, has been a major goal for some years.Much attention has concentrated on the use of viruses as deliveryvehicles, for example adenoviruses for epithelial cells in therespiratory tract with a view to corrective gene therapy for cysticfibrosis (CF). However, despite some evidence of successful genetransfer in CF patients, the adenovirus route remains problematic due toinflammatory side-effects and limited transient expression of thetransferred gene. Several alternative methods for in vivo gene deliveryhave been investigated, including studies using cationic surfactants.Gao, X et al. (1995) Gene Ther. 2, 710-722 demonstrated the feasibilityof this approach with a normal human gene for CF transmembraneconductance regulator (CFTR) into the respiratory epithelium of CF miceusing amine carrying cationic lipids. This group followed up with aliposomal CF gene therapy trial which, although only partiallysuccessful, demonstrated the potential for this approach in humans(Caplen, N J. et al., Nature Medicine, 1, 39-46, 1995). More recentlyother groups have investigated the potential of other cationic lipidsfor gene delivery, for example cholesterol derivatives (Oudrhiri, N etal. Proc.Natl.Acad. Sci. 94, 1651-1656, 1997). This limited studydemonstrated the ability of these cholesterol based compounds tofacilitate the transfer of genes into epithelial cells both in vitro andin vivo, thereby lending support to the validity of this generalapproach.

[0005] These studies, and others, show that in this new field ofresearch there is a continuing need to develop novel low-toxicitysurfactant molecules to facilitate the effective transfer ofpolynucleotides into cells both in vitro for transfection in cell-basedexperimentation and in vivo for gene therapy and antisense treatments.The present invention seeks to overcome the difficulties exhibited byexisting compounds.

[0006] The invention relates to the peptide-based gemini compoundscomprising two linked chains:

[0007] each chain having:

[0008] (1) a positively charged hydrophilic head, Q¹ or Q², formed fromone or more amino acids and/or amines;

[0009] (2) a central portion, P¹ or P², having a polypeptide backbone;and

[0010] (3) a hydrophobic tail, R¹ or R²;

[0011] the central sections of each chain being linked together bybridge Y through residues in P¹ and P².

[0012] Preferably the central portion is made up of two or three aminoacids, P^(a) (optional), P^(b) and P^(c), in which:

[0013] P^(a) is a D- or L-amino acid, preferably hydrophilic, such asthreonine or serine,

[0014] P^(b) is preferably D- or L-cysteine, serine or threonine, and

[0015] P^(c) is preferably D- or L-serine or threonine and is linked toR¹ or R².

[0016] Preferred compounds of the present invention include compounds ofthe formula (I):

[0017] where:

[0018] A¹ and A⁵ ,which may be the same or different, is a positivelycharged group formed from one or more amino acids or amines joinedtogether in a linear or branched manner and preferably bonded by anamide (CONH) bond;

[0019] A²/A⁶CH(NH)CO, which may be the same or different, is derivedfrom an amino acid, preferably serine;

[0020] p and q, which may be the same or different, is 0 or 1;

[0021] X¹/X²CH₂CH(NH)CO, which may be the same or different, is derivedfrom cysteine (X¹/X²═S), serine or threonine (X¹/X²═O);

[0022] A⁴/A⁸CH(NH)CO, which may be the same or different, is derivedfrom serine or threonine;

[0023] Y is a linker group, preferably (CH₂)_(m) where m is an integerfrom 1 to 6, most preferably 2, and may be a disulphide bond when X¹ andX² is each S;

[0024] R¹ and R² are C₍₁₀₋₂₀₎ saturated or unsaturated alkyl groups, andW and Z are NH, O, CH₂ or S; or

[0025] a salt, preferably a pharmaceutically acceptable salt thereof.

[0026] Preferably, the compound is symmmetrical, that is A¹ and A⁵ arethe same, A² and A⁶ are the same, A⁴ and A⁸ are the same, R¹ and R² arethe same, and W and Z are the same.

[0027] Representative examples of A¹/A⁵ include D- or L-amino acidsselected from arginine, lysine, ornithine and histidine, preferablylysine, or amines such as spermine and spermidine. Up to seven aminoacids and /or amines may be linked in a linear or branched chain.Prefered examples include groups having two or three lysines orornithines or a combination of lysine, ornithine, arginine andhistidine, for instance:

COCH(NHR)(CH₂)₄NHCO(NH₂)(CH₂)₄NH₂

[0028] or

COCH(NHR)(CH₂)₃NHCO(NH₂)(CH₂)₃NH₂

[0029] or

COCH(NHR)(CH₂)₄NHCO(NH₂)(CH₂)₃NH₂

[0030] in which R is H or NHCO(NH₂)(CH₂)₄NH₂ or NHCO(NH₂)(CH₂)₃NH₂

[0031] Preferably, —X¹—Y—X²— is —SCH₂CH₂S— or —OCH₂CH₂O—

[0032] Preferably, R¹ and R² is each a C₁₂-C₂₀ alkyl group, for instanceC₁₂.

[0033] Preferably, W and Z is NH, thereby forming a further amide (CONH)bond.

[0034] Compounds of the present invention may be prepared from readilyavailable starting materials using synthetic peptide chemistry wellknown to the skilled person. For prefered compounds of the presentinvention a useful intermediate is the compound:

[0035] which is synthesised in a multi-stage process beginning, forinstance, with the construction of the di-cysteine part and subsequentlybuilding up the hydrophilic head by attaching a serine moiety at thecarboxyl group of each cysteine moiety, using standard peptidechemistry, and then attaching the hydrocarbon chains to the carboxylgroup of the serine moiety using a standard an-ide forming reaction wellknown to those skilled in the art. This intermediate can then be takenthrough to compounds of formula (I) by further reaction at the nitrogensof the cysteine residues.

[0036] Another aspect of the invention relates to methods for using thepeptide-based gemini compounds. Such uses include facilitating thetransfer of oligonucleotides and polynucleotides into cells forantisense, gene therapy and genetic immunisation (for the generation ofantibodies) in whole organisms. Other uses include employing thecompounds of the invention to facilitate the transfection ofpolynucleotides into cells in culture when such transfer is required,in, for example, gene expression studies and antisense controlexperiments among others. The polynucleotides can be mixed with thecompounds, added to the cells and incubated to allow polynucleotideuptake. After further incubation the cells can be assayed for thephenotypic trait afforded by the transfected DNA, or the levels of mRNAexpressed from said DNA can be determined by Northern blotting or byusing PCR-based quantitation methods for example the Taqman® method(Perkin Elmer, Connecticut, USA). Compounds of the invention offer asignificant improvement, typically between 3 and 6 fold, in theefficiency of cellular uptake of DNA in cells in culture, compared withcompounds in the previous art. In the transfection protocol, the geminicompound may be used in combination with one or more supplements toincrease the efficiency of transfection. Such supplements may beselected from, for example:

[0037] (i) a neutral carrier, for example dioleylphosphatidylethanolamine (DOPE) (Farhood, H., et al (1985) Biochim.Biophys. Acta 1235 289);

[0038] (ii) a complexing reagent, for example the commercially availablePLUS reagent (Life Technologies Inc. Maryland, USA) or peptides, such aspolylysine or polyornithine peptides or peptides comprising primarily,but not exclusively, basic amino acids such as lysine, ornithine and/orarginine. The list above is not intended to be exhaustive and othersupplements that increase the efficiency of transfection are taken tofall within the scope of the invention.

[0039] In still another aspect, the invention relates to the transfer ofgenetic material in gene therapy using the compounds of the invention.

[0040] Yet another aspect of the invention relates to methods to effectthe delivery of non-nucleotide based drug compounds into cells in vitroand in vivo using the compounds of the invention.

[0041] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0042] “Amino acid” refers to dipolar ions (zwitterions) of the form+H₃NCH(R)CO₂—. They are differentiated by the nature of the group R, andwhen R is different from hydrogen can also be asymmetric, forming D andL families. There are 20 naturally occurring amino acids where the Rgroup can be, for example, non-polar (e.g. alanine, leucine,phenylalanine) or polar (e.g. glutamic acid, histidine, arginine andlysine). In the case of un-natural amino acids R can be any other groupwhich is not found in the amino acids found in nature.

[0043] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications have been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0044] “Transfection” refers to the introduction of polynucleotides intocells in culture using methods involving the modification of the cellmembrane either by chemical or physical means. Such methods aredescribed in, for example, Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). The polynucleotides may be linear orcircular, single-stranded or double-stranded and may include elementscontrolling replication of the polynucleotide or expression ofhomologous or heterologous genes which may comprise part of thepolynucleotide.

[0045] The invention will now be described by way of the followingdescriptions and examples.

[0046] Descriptions

[0047] Description 1. Bisthioether 3

[0048] A IL 3-necked flask equipped with mechanical stirrer, refluxcondenser and dropping funnel was flushed with N₂ directly into theflask through the condenser. A solution of 31.3 g (0.20 mole)L-cysteine.hydrochloride.xH₂O (1) in 100 ml (degassed ultrasonic for 10minutes) was added to the flask. A degassed solution of 34 g (0.40 mole)NaHCO₃ in 300 ml H₂O was added, followed by the dropwise addition (30minutes) of a degassed solution of 18.8 g (8.6 ml; 0.10 mole)1,2-dibromoethane (2) in 100m] EtOH. After another 30 minutes themixture was heated to 65-70° C. and stirred, still under N₂, for another3 hours (within 1 minutes precipitation started). The mixture was cooledto 20° C., filtered, rinsed with 30 ml H₂O and with 100 ml acetone (2×).After drying 19.4 g white solid was obtained which still contained somefree cysteine (¹H NMR). The solid was suspended in 250 ml 2.5% NH₄OH and25% NH₄OH was added until a clear solution was obtained. To thissolution 15 mg KCN was added and the mixture was stirred for 30 minutes.The solution was acidified to pH 6 using HOAc and stirred for 30 minuteswhilst cooling to 5° C. The solid was collected, rinsed with H₂O (100ml), acetone (2×100 ml) and dried, yielding 18.1 g (68%) 3 as a whitesolid.

[0049] Description 2. Boc-L-Leucine 5

[0050] 19.7 g (0.15 mole) L-Leucine 4 was suspended in 200 ml H₂O and6.75 g (0.17 mole) NaOH was added. The clear solution was cooled to <10°C. and a solution of 36 g (0.165 mole) (BOC)₂O in 100 ml THF was addeddropwise keeping T<10° C. (30 minutes). After stirring for 4 hours atroom temperature the mixture was acidified to pH 2 by adding 1N HCl. Themixture was extracted with EtOAc (250, 100 and 100 ml), the combinedorganic layers were dried on Na₂SO₄ and evaporated, yielding 40 g(>100%) 5 as a colorless oil which contained some THF but was used assuch.

[0051] Description 3. Boc-L-Leucine-OSuc 7

[0052] 40 g crude 5 (max. 0.15 mole) was dissolved in 400 ml THF(distilled prior to use from LAH) under N₂. After addition of 17.3 g(0.15 mole) N-hydroxysuccinimide (6) and 30.9 g (0.15 mole) DCC themixture was stirred for 3.5 hours at room temperature. The mixture wasfiltered over a P₂ glassfilter, the filter was rinsed with 50 ml THF andthe filtrate was evaporated. The residue was dissolved in 400 mlrefluxing isopropylether, the solution was filtered hot and the filtratewas placed at 4° C. for 20 hours. The solid was collected, rinsed with50 ml IPE and dried, yielding 32 g (65%) 7 as a white solid.

[0053] Description 4. Compound 8

[0054] 8.05 g (30mmole) 3 was suspended in 200 ml H₂O and after additionof 8.3 g (60 mmole) K₂CO₃ the mixture was heated to get a clearsolution. After cooling to room temperature a solution of 19.7 g (60mmole) 7 in 200 ml THF was added at once. The mixture was stirred atroom temperature for 20 hours, followed by acidification to pH 6 (30%HCl). After filtration the filtrate was acidified to pH 2-3 (30% HCl)and extracted with CHCl₃ (250, 100 and 100 ml). The combined organiclayers were once washed with brine (50 ml), dried on Na₂SO₄ andevaporated, yielding 23 g crude 8 as a white foam which was used assuch.

[0055] Description 5. Compound 9

[0056] 23 g crude 8 was dissolved in 200 ml EtOAc, cooled to 0° C. andHCl-gas was bubbled through for 1 hour, followed by stirring at thistemperature for another hour. The solid was collected, rinsed with etherand dried under vacuo over KOH. This yielded 15.4 g (91%) 9 as ahygroscopic nearly white solid.

[0057] Description 6. Compound 10

[0058] 17.0 g (30 mmole) 9 dissolved in 250 ml H₂O and after cooling to<10° C. in an ice/waterbath 7.2 g (180 mmole) NaOH was added. Afterstirring for 10 minutes at room temperature a solution of 14.4 g (66mmole) lauroylchloride in 50 ml THF was added dropwise in 5 minutes. Themixture was stirred for 20 hours and extracted with hexame (2×150 ml). A3-layer system formed and the lower 2 layers were acidified to pH 1-2 (1N HCl) and extracted with ether (200, 100, 100 and 50 ml). The combinedether layers were dried on MgSO4 and evaporated, yielding 24 g (93%) 10as an oil/foam. This material was used as such.

[0059] Description 7. Compound 11

[0060] 24 g (max 27.5 mmole) 10 dissolved in 400 ml THF (distilled fromLAH) under N₂. After addition of 6.33 g (55 mmole) N-hydroxysuccinimide(6) and 11.33 g (55 mmole) DCC the mixture was stirred at roomtemperature for 20 hours. The mixture was filtered over a large P₂glassfilter, another 250 ml THF was added to speed up filtration. Thefilter was rinsed with another 100 ml THF. The filtrate was evaporatedyielding 40 g white solid and this crude material was recristallizedfrom 400 ml IPA. Stirred 1 hour 0° C. and collected. After drying 22.3 g(77%) 11 was obtained as white solid.

[0061] Description 8. Compound 13

[0062] 542 mg (2.2 mmole) H.Arg.NH₂.2HCl (12) was dissolved in 15 ml H₂Oand 304 mg (2.2 mmole) K₂CO₃ was added. A solution of 1.05 g (1.0 mmole)11 in 15 ml THF was added at once and the mixture was stirred at roomtemperature for 20 hours. Most of the THF was evaporated and an oilformed in the waterlayer. This suspension was extracted with ether (6×50ml), the combined ether layers were dried on MgSO₄ and evaporated,yielding 9500 mg (82%) 13 free amine as a yellow solid. Treatment ofthis material with HCl-gas in EtOAc/CH₂C. 12 gave 13 as a yellow solid.

[0063] Description 9. Compound 15

[0064] 5.25 g (5.0 mmole) 11 dissolved in 50 ml THF (some heating wasneeded) and a solution of 1.31 g (10.5 mmole) Taurine (14) and 1.45 g(10.5 mmole) K₂CO₃ in 50 ml H2O was added at once. After stirring atroom temperature for 20 hours most of the THF was removed by evaporationand 300 ml MeOH was added. Mixture placed at −20° C. for 20 hours, solidcollected, rinsed with MeOH and dried. This yielded 3.3 g 15contaminated with N-hydroxysuccinimide., which was combined with 700 mgimpure material of an other run. This 4.0 g was recrystallized from 200ml MeOH+50 ml H₂O. Some solid was removed by filtration and the clearfiltrate was placed at −20° C. for 2 hours, the solid was colected,rinsed with MeOH and dried. This yielded 2.0 g 15 as an off white solid.A 2^(nd) crop of 800 mg was obtained from the filtrate.

[0065] Description 10. Compound 16

[0066] 1.05 g (1.0 mmole) 11 was dissolved in 20 ml THF and a solutionof 275 mg (2.2 mmole) 2-aminoethylphosphonic acid and 300 mg (2.2 mmole)K₂CO₃ in 20 ml H₂O was added at once. After stirring at room temperaturefor 20 hours most of the THF was removed by evaporation and the aqueoussolution was freeze dried. This yielded a white solit which wasrecrystallized from 20 ml MeOH and placed at −20° C. for 20 hours. Thesolid was collected and dried yielding 150 mg. ¹H NMR showed it to beN-hydroxysuccinimide. The filtrate was evaporated and the remainingyellow oil was dissolved in 10 ml refluxing MeOH and after addition of20 ml IPA placed at −20° C. for 4 hours. Solid was collected and dried,yielding 260 mg.

[0067] The filtrate was evaporated, dissolved in 20ml EtOAc and placedat −20° C. for 20 hours. The solid was collected.

[0068] Description 11. Compound 17

[0069] 1.05 g (1.0 mmole) 11 was dissolved in 20 ml THF and a solutionof 310 mg (2.2 mmole) O-phosphocolamine and 300 mg (2.2 mmole) K₂CO₂ in20 ml H₂O was added at once. After stirring at room temperature for 20hours most of the THF was removed by evaporation and the aqueoussolution was freeze dried. The white solid was recrystallized from 20 mlMeOH and placed at −20° C. for 20 hours. The solid was collected, rinsedwith MeOH and dried. This yielded 310 mg white solid. No product.

[0070] The filtrate was evaporated and the remaining yellow solid wasdissolved in 10 ml MeOH, 20 ml IPA was added and the mixture was placedat −20° C. for 20 hours. Solid collected, 20-30 mg

[0071] Filtrate evaporated, residue dissolved in 25 ml EtOAc, placed at−20° C. and after 20 hours the solid was collected.

[0072] Description 12. Boc-Glycine Boc-Glycine 19

[0073] 18.8 g (0.25 mole) Glycine 18 was suspended in 250 ml H₂O and 11g (0.275 mole) NaOH was added. The clear solution was cooled to <10° C.and a solution of 60 g (0.165 mole) (BOC)₂O in 250 ml THF was addeddropwise keeping T<10° C. (20 minutes). After stirring for 20 hours atroom temperature the mixture was acidified to pH 1 by adding 1N HCl. Themixture was extracted with EtOAc (250, 100 and 100 ml), the combinedorganic layers were dried on MgSO₄ and evaporated, yielding 47.5 g(>100%) 5 as a colourless oil which contained some THF but was used assuch.

[0074] Description 13. Boc-Glycine-OSuc 20

[0075] 47.5 g crude 19 (max. 0.25 mole) was dissolved in 500 ml THF(distilled prior to use from LAH) under N₂. After addition of 30 g (0.26mole) N-hydroxysuccinimide (6) and 53.5 g (0.26 mole) DCC at <10° C.,the mixture was stirred for 20 hours at room temperature. Now themixture was filtered over 1 cm celite on a P2 glassfilter, the filterwas rinsed with 200 ml THF and the filtrate was evaporated. The crudematerial (56 g) was recrystallized from refluxing isopropylether/THF(600 ml 1:1), the solution was filtered hot and the filtrate was stirredat 0° C. for 3 hours. The solid was collected, rinsed with 50 ml IPE anddried, yielding 16.4 g (24%) 20 as a white solid. Filtrate evaporatedand stored.

[0076] Description 14. Compound 21

[0077] 8.05 g (30 mmole) 3 was suspended in 200 ml H₂O and after addtionof 8.3 g (60 mmole) K₂CO₃ the mixture was heated to get a clearsolution. After cooling to <40° C. a solution of 16.3 g (60 mmole) 20 in200 ml THF was added in 4 portions within 2 minutes. The mixture wasstirred at room temperature for 72 hours, followed by acidification topH 6 (30% HCl). After filtration the filtrate was acidified to pH 2-3(30% HCl) and extracted with CHCl₃ (250, 100 and 100 ml). The combinedorganic layers were once washed with brine (50 ml), dried on MgSO₄ andevaporated, yielding 15.8 g (90%) 21 as a white foam which was used assuch.

[0078] Description 15. Compound 22

[0079] 15.8 g (27 mmole) crude 21 was dissolved in 300 ml EtOAc andHCl-gas was bubbled through for 1 hour, followed by stirring at 0° C. inan ice/waterbath and a solution of 12.2 g (56 mmole) lauroylchloride in50 ml THF was added at once. The mixture was stirred for 20 hours andextracted with hexane (2×100 ml). The water layer was acidified to pH1-2 (1 N HCl) and extracted with ether (3×150 ml). A solid formed duringextraction, which was collected and dried. This yielded 7.5 g (39%) 23white solid.

[0080] The filtrate was evaporated and the remaining slurry was stirredin 250 ml Et₂O. An attempt to collect the solid failed and addition of25 ml MeOH gave a clear solution. This solution was placed at −20° C.for 20 hours. The solid was collected, rinsed with ether and dried.

[0081] Description 16. Compound 24

[0082] 7.47 g (10 mmole) 23 dissolved in 200 ml THF (distilled from LAH)under N₂. After addition of 2.53 g (22 mmole) N-hydroxysuccinimide (6)and 4.53 g (22 mmole) DCC the mixture was stirred at room temperaturefor 72 hours. The mixture was filtered over a 1 cm layer celite on alarge P₂ glassfilter (very slow). The filtrate was evaporated yielding1.90 g 24 as a foam.

[0083] The filer was rinsed with 300 ml dioxane and the filtrate wasevaporated yielding 6.9 g 24 as a foam. Total yield 8.8 g (94%).

[0084] Description 17. Compound 25

[0085] 542 mg (2.2 mmole) H.Arg.NH₂.2HCl (12) was dissolved in 15 ml H₂Oand 750 mg (5.4 mmole) K₂CO₃ was added. A solution of 941 mg (1.0 mmole)24 in 15 ml THF was added at once and the mixtrue was stirred at roomtemperature for 20 hours. Most of the THF was evaporated and thewaterlayer was extracted with EtOAc (2×50 ml), the combined EtOAc layerswere dried on MgSO₄ and evaporated, yielding 150 mg 25 free amine as afoam. Both portions were combined and dissolved in CH₂Cl₂ and after theaddition of 75 ml EtOAc, HCl-gas was bubbled through for 1.5 hour. Nowthe mixture was cooled to 0° C. and stirred for another 2 hours.Attempts to collect the solid failed, so 100 ml ether was added and themixture was stirred at room temperature for 20 hours. The solid wascollected, rinsed with ehter and dried, yielding 520 mg 25 as a slightlybrown solid.

[0086] Description 18. Compound 26

[0087] 250 mg (2.0 mmole) taurine (14) was dissolved in 15 ml H₂O and280 mg (2.0 mmole) K₂CO₃ was added. Now a solution of 941 mg (1.0 mmole)24 in 15 ml THF was added at once and the mixture was stirred at roomtemperature for 20 hours. Most of the THF was evaporated and the aqueoussolution was freeze dried. The resulting white solid was recrystallizedfrom 30 ml MeOH, stirred 3 hours at 0° C. and the solid was collected,rinsed with ether and dried.

[0088] This yielded 225 mg 26 as a white solid.

[0089] The filtrate was partly evaporated and placed at −20° C. for 20hours. The solid was collected, rinsed with ether and dried, yieleing210 mg 26 as a white solid. Both portions were combined.

[0090] Description 19. Compound 27

[0091] 26.8 g (0.1 mole) 3 was suspended in 300 ml H₂O and 9.6 g (0.24mole) NaOH was added. A clear solution formed within 5 minutes, themixture was cooled to <10° C. and a solution of 43.6 g (0.2 mole) BOC₂Oin 300 ml THF was added dropwise in 30 minutes. The mixture was stirredat room temperature overnight. After addition of a solution of 2.5 g(0.06 mole) NaOH in 25 ml H₂O and 15 g (0.07 mole) BOC₂O in 75 ml THFthe mixture was stirred for another 18 hours.

[0092] The mixtue was acidified to pH 2 by adding 2N HCl and afteraddition of 300 ml brine, extracted with THF (3×400 ml) and EtOAc (2×300ml) The combined organic layers were dried on MgSO₄ and evaporated,yielding 42 g white solid. This solid was recrystallized fromMEK/pentane, stirred at room temperature for 2 hours and placed at −20°C. for 2 hours. The solid was collected and dried, yielding 37.8 g (81%)27 as a white solid.

[0093] Description 20. Compound 28

[0094] 8.9 g (19 mmole) 27 was dissolved in 300 ml THF (from LAH) underN₂ and 4.37 g (38 mmole) 6 and 7.38 g (38 mmole) DCC were added. Afterstirring at room temperature for 18 hours the mixture was filtered over1 cm celite, the filter was rinsed with another 300 ml THF and thefiltrate was evaporated yielding 11.1 g (88%) 28 as a white solid.

[0095] Description 21. Compound 29

[0096] 2.7 g (20.6 mmole) L-leusine (4) and 2.8 g (20.3 mmole) K₂CO₂were dissolved in 100 ml H₂O and a suspension of 6.6 g (10 mmole) 28 in50 ml dioxane was added. The mixture was stirred at room temperature for20 hours and most of the dioane was removed by evaporation. The aqueoussolution was extracted with 50 ml ether and acidified to pH 1 byaddition of 30% HCl. Now the mixture was extracted with CHCl₃ (150, 100and 50 ml), the combined organic layers were washed with brine (200 ml),dried on MsSO4 and evaporated, yielding a white foam which was strippedwith THF to get 7.5 g (>100%) 29 as a solid white foam.

[0097] Description 22. Compound 30

[0098] 7.5 g crude 29 (max. 10 mmole) was dissolved in 100 ml THF (fromLAH) under N₂, 2.3 g (20 mmole) N-hydroxysuccinimide (6) and 4.12 g (20mmole) DCC were added and the mixture was stirred at room temperaturefor 20 hours. The mixture was filtered over 1 cm celite, the filter wasrinsed with THF and the filtrate was evaporated, yielding 9.5 g whitefoam which was recrystallized from 75 ml IPA and placed at −20° C. for 3hours. The solid was collected but liquified immediately on the glassfilter. The oily material was dissolved in 20 ml THF and evaporated,yielding 6.5 g (73%) 30 as a solid white foam.

[0099] Description 23. Compound 31

[0100] 6.5 g (7.3 mmole) 30 was dissolved in 100 ml THF and afteraddition of 2.78 g (15 mmole) dodecylamine the mixture was stirred atroom temperature for 18 hours. After evaporation a foam was obtainedwhich was dissolved in 100 ml CHCl₃. The solution was washed with H₂O(2×75 ml), dried on MgSO4 and evaporated, yielding 7.5 g (100%) 31 as asolid foam.

[0101] Description 24. Compound 32

[0102] 7.5 g (7.3 mmole) crude 31 was dissolved in 250 ml EtOAc underheating and after cooling to room temperature HCl-gas was bubbledthrough for 2 hours. Stirring was continued at 0° C. for 3 hours. Thesolid was collected, rinsed with ether and dried under vacuo over KOH.This yielded 4.0 g (60%) 32 as a white solid.

[0103] Description 25. Compound 33

[0104] 903 mg (1.0 mmole) 32 was dissolved in 10 ml H₂O under heating(gel formed), after cooling to room temperature, 80 mg (2.0 mmole) NaOHdissolved in 2 ml H₂O was added. A suspension was formed and THF wasadded until a clear solution was obtained. Now a solution of 572 mg (2mmole) BOC-β-alaOSuc (42) in 5 ml THF was added and the mixture wasstirred at room temperature for 5 hours. Most of the THF was removed byevaporation, another 30 ml H₂O was added and after stirring for anotherhour the solid was collected, rinsed with 10 ml H₂O and dried. Thisyielded 1.0 g (85%) 33 as an off white solid.

[0105] Description 26. Compound 34

[0106] 1.0 g (0.85 mmole) 33 suspended in 25 ml EtOAc and 25 ml CH₂Cl₂added to get a clear solution. HCl-gas bubbled through for 1.5 hour andstirred at 0° C. for another 2 hours. No solid had formed so most of theCH₂Cl₂ was removed by evaporation and stirring at 0° C. was continuedfor anothe 30 minutes. The solid was collected, partly by filtration(very slow), mainly be centrifugation. Total yield after drying 810 mg(91%) 34 as a yellow solid.

[0107] Description 27. Compound 36

[0108] 4.2 g (40 mmole) L-serine 35 and 5.53 (40 mmole) K₂CO₃ weredisslved n 300 ml H₂O and a suspension of 12.8 g (max. 19 mmole) 28 in300 ml THF was added. The mixture was stirred at room temperature for 72hours and most of the THF was removed by evaporation. The aqueoussolution was acidified to pH 1 by addition of 1N HCl. The mixture wasextracted with CH₂Cl₂+15% MeOH (250, 100 and 100 ml), the combinedorganic layers were dried on MgSO₄ and evaporated, yielding 8.5 g (70%)36 as a white solid foam which was used as such.

[0109] Description 28. Compound 37

[0110] 8.5 g (max. 13.2 mmole) 36 was dissolved in 200 ml THF (from LAH)under N₂ and after addition of 3.46 g (30 mmole) N-hydroxysuccinimide(6) and 6.2 g (30 mmole) DCC the mixture was stirred at room temperaturefor 24 hours. The mixture was filtered over 1 cm celite, the filter wasrinsed with 50 ml THF and evaporated. This yielded 12.5 g (>100%) 37 asa white foam which was used as such.

[0111] Description 29. Compound 38

[0112] 12.5 g crude (max 13.2 mmole) 37 was dissolved in 200 ml THF andstirred with 5.0 g (27 mmole) dodecylamine at room temperature for 48hours. The THF was removed by evaporation and the residue was dissolvedin 250 ml CHCl₃ and extracted with brine (2×150 ml). The combined brinelayers were extracted with 50 ml CHCl₃ and the combined CHCl₃ layerswere dried on MgSO₄ and evaporated. This yielded 15.4 g (>100%) 38 as anearly white solid which was used as such.

EXAMPLES Example 1

[0113] Compound 392-amino-3-{2-[2-amino-2-(1-dodecylcarbamoyl-2-hydroxy-ethylcarbamoyl)-ethylsulphanyl]-ethylsulphonyl}-N-(1-dodecylcarbamoyl-2-hydroxy-ethyl-)-propionamide

[0114] 15.4 g (max. 13.2 mmole) 38 dissolved in 400 ml EtOAc and HCl-gaswas bubbled through for 1.5 hour. The mixture was stirred at 0° C. for 2hours, the solid was collected, rinsed with ether and dried, yielding9.9 g (88%) 39 as a white solid.

Example 2

[0115] Compound 40

[0116] 4.25 g (5 mmole) 39 was dissolved in 100 ml H₂O with heating andafter cooling to <40° C. a solution of 460 mg (10 mmole) NaOH in 10 mlH₂O was added. A suspension formed and THF was added until a clearsolution was obtained (150 ml). Next 2.86 g (10 mmole) BOC-β-alaOSu (42)was added and the mixture was stirred at room temperature for 20 hours.Most of THF was removed by evaporation, another 100 ml H₂O was added andthe mixture was stirred at 0° C. for 3 hours. The solid was collected,rinsed with 20 ml H₂O and dried. This yielded 5.2 g (93%) 40 as a nearlywhite solid.

Example 3

[0117] Compound 41

[0118] 5.2 g (4.6mmole) 40 was dissolved in 100 ml CH₂Cl₂ and 200 mlEtOAc was added. HCl-gas was bubbled through for 1.5 hour and stirringwas continued at 0° C. for 2 hours. The solid was collected, rinsed withether and dried, yielding 4.7 g (100%) 41 as off white solid.

Example 4

[0119] Compounds 42 and 43

[0120] After neutralization of compounds 34 and 41 using 2 eq. of NaOHin MeOH both compounds were treated with (CH₂O)_(n) and NaCNBH₃ under N₂for 18 hours. In both reactions complex mixtures were formed, probablydue to alkylation on amide nitrogen as well.

Example 5

[0121] Compound 44

[0122] 332 mg (0.39 mmole) 39 was dissolved in 15 ml H₂O under heatingand after cooling to <40° C. a solution of 33 mg (0.83 mmole) NaOH in 1ml H₂O was added. A white suspension formed and THF was added until aclear solution was obtained (25 ml). To this solution 499 mg (0.78mmole) BOC-Arg(Z)₂—OSu (47) was added and the mixture was stirred atroom temperature for 20 hours. Most of the THF was evaporated andanother 15 ml H₂O was added. After 2 hours stirring the solid wascollected, rinsed with H₂O and dried, yielding 700 mg (98%) 44 as awhite solid.

Example 6

[0123] Compound 45

[0124] 100 mg (0.05 mmole) 44 was dissolved in 20 ml HOAc and 500 mg 10%Pd on Carbon (0.5 mmole Pd) was added. The mixture was stirred under H₂(5 bar) for 48 hours. The mixture was filtered over 1 cm celite, thefilter was rinsed with 10 ml HOAc and the filtrate was evaporated. Thisyielded 100 mg crude 45 as a green oil.

Example 7

[0125] Compound 46

[0126] 100 mg crude 45 (max 0.05 mmole) was disslved in 10 ml CH₂Cl₂ and10 ml EtOAc was added. HCl-gas was bubbled through for 1 hour and themixture was stirred 18 hours at room temperature. Most of the CH₂Cl₂ wasremoved by evaporation, 30 ml ether was added and the mixture wasstirred at 0° C. for 1 hour. No crystalline material had formed so themixture was evaporated, yielding 75 mg crude 46 as a yellow oil.

Example 8

[0127] Compound 49

[0128] 850 mg (1.0 mmole) 39 was dissolved in 20 ml H₂O and after 88 mg(2.2 mmole) NaOH was added a suspension formed. Now THF was added untila clear solution was obtained (30 ml) and 974 mg (2.2 mmole) BOC₂LysOSuc(compound 48) was added. After stirring at room temperature for 20 hoursmost of the THF was removed by evaporation, another 20 ml H₂O was addedand the mixtrue was stirred for 2 hours. The solid was collected, rinsedwith H₂O and dried, yielding 1.35 g (90%) 49 as a white solid.

Example 9

[0129] Compound 50

[0130] 500 mg 49 was dissolved in 25 ml CH₂Cl₂ and after addition of 25ml EtOAc HCl-gas was bubbled through for 1 hour, the mixture was stirredat 0° C. for 1.5 hour. An attempt to collect the solid failed, 40 mlether was added and stirring was continued for 18 hours. The solid wascollected, rinsed with ehter and dried, yielding 290 mg 50 as a whitesolid.

Example 10

[0131] Compound 51

[0132] 425 mg (0.43 mmole) 41 was dissolved in 20 ml H₂O and 44 mg (1.1mmole) NaOH was added. A suspension formed and THF was added until aclear solution was obtained (25 ml). After addition of 487 mg (1.1mmole) 48 the mixture was stirred at room temperature for 20 hours. Mostof the THF was evaporated, another 20 ml H₂O was added and afterstirring for 1.5 hour the solid was collected, rinsed with H₂O anddried. This yielded 750 mg 51 as a white solid.

Example 11

[0133] Compound 52

[0134] 250 mg 51 was dissolved in 30 ml CH₂Cl₂ and after addition of 30ml EtOAc, HCl-gas was bubbled through for 1 hour, the mixture wasstirred at 0° C. for 1.5 hour. The solid was collected, rinsed withether and dried, yielding 120 mg 52 as a white salt.

Example 12

[0135] Compound 57 (SucOSerLysBOC₂)

[0136] 4.43 g (10 mmole) BOC₂LysOSuc (48) was dissolved in 50 ml THF anda solution of 1.16 g (11 mmole) L-serine and 1.52 g (11 mmole) K₂CO₃ in50 ml H₂O was added immediately. The mixture was stirred at roomtemperature for 72 hours. Most of the THF was removed by evaporation andthe remaining slurry was acidified to pH 2 by the addition of 1M HCl andextracted with CHCl₂ (2×75 ml). The combined organic layers were dried(Na₂SO₄) and evaporated, yielding 57 as a white solid foam which wasused as such in example 43.

Example 13

[0137] Compound 54

[0138] 850 mg (1.0 mmole) 39 was dissolved in 30 ml H₂O and 88 mg (2.2mmole) NaOH was added, followed by the addition of 30 ml THF to get aclear solution. A solution of 1.5 g (max. 2.3 mmole) 57 in 30 ml THF wasadded immediately and the solution was stirred at room temperature for48 hours. Most of the THF was removed by evaporation, another 30 ml H₂Owas added and stirring was continued for 1 hour. Because no solid hadformed, the mixture was extracted twice with 75 ml EtOAc/ether (2:1).The combined organic layers were dried (Na₂SO₄) and evaporated yieldingthe BOC-protected intermediate as a solid foam.

[0139] This foam was dissolved in 25 ml CH₂Cl₂ and 50 ml EtOAc wasadded. HCl gas was bubbled through the clear solution for 1 hour andstirring was continued at 0° C. for another hour. The salt wascollected, rinsed with ether and dried under vacuum, yielding 1.15 g(85%) 54 as a slightly brown solid.

Example 14

[0140] Compound 55

[0141] 1.18 g (1.0 mmole) 50 was dissolved in 25 ml H₂O and 176 mg (4.4mmole) NaOH was added, followed by the addition of 30 ml THF to get aclear solution. 974 mg (2.2 mmole) 48 were added and the mixture stirredat room temperature for 48 hours. Most of the THF was removed byevaporation, another 50 ml H₂O was added and the mixture was stirred for2 hours. Because no solid was formed the mixture was extracted withether (2×100 ml), the combined organic layers were dried (Na₂SO₄) andevaporated, yielding 2 g of the BOC-protected intermediate as a solidfoam. The foam was dissolved in 50 ml EtOAc and HCl-gas was bubbledthrough the solution for 1 hour and stirring was continued at 0° C. foranother hour. The salt was collected, rinsed with ether and dried undervacuum, yielding 1.15 g (76%) 55 as a nearly white solid.

Example 15

[0142] Compound 56

[0143] Compound 56 was synthesised as for compound 55 except that 1.95 g(4.4 mmole) 48 was used. This yielded 1.1 g (60%) 56 as an off whitesolid.

Example 16

[0144] Compounds 57, 58 and 59

[0145] Compounds 57, 58 and 59 are synthesised in a similar manner tothe compounds described above. Compound 39, or an intermediateequivalent to compound 39 but having different saturated or unsaturatedhydrocarbon chains, is combined with an ornithine compound usingsynthetic peptide chemistry well known to the skilled person.

[0146] It will be appreciated by a person skilled in the art that in theformulae shown in the examples above, the hydrogen atoms have beenomitted from the N, C and O atoms, where appropriate, for clarity.

Example 17

[0147] Transfection of Recombinant Plasmid Expressing Luciferase intoHEK293 Cells Using Peptide-based Gemini Compounds.

[0148] All tissue culture reagents were obtained from Life TechnologiesInc. HEK 293 cells were seeded at 2-3×10⁵ cells per well in Nuncsix-well culture plates, 24 hours prior to transfection. The cells wereseeded in 2 mls Dulbeccos Modified Eagle medium containing Earles saltsand supplemented with 10% v/v foetal bovine serum (=complete medium).The cells were grown at 37° C. in 5% CO₂ in a humidified atmosphere. 6ug DNA (“luciferase control plasmid” from Promega Corp.) were dissolvedin 100 ul serum free medium (OPTI-MEMO). The peptide-based geminicompounds were made up at 1 mg/ml in tissue culture grade water and thendiluted in OPTI-MEM® to the appropriate concentration to a final volumeof 100 ul. The DNA and gemini solutions were mixed (to a total volume of200 ul; final concentrations of 5, 25, 50, 100, 150, 200, 250 and 300ug/ml) and left at room temperature for 15 minutes. The DNA/gemini mixwas placed onto the cells in each well and left in contact for 18-20hours. The cells were then washed twice with phophate buffered salineprior to 1 ml of fresh complete medium being added. Cells were incubatedfor a further 24 hours prior to lysis and luciferase activity assayed.

[0149] All luciferase activity assays were performed using the CanberraPackard (Berkshire, UK) Luclite kit according to the manufacturer'sinstructions with the exception that the cells in each well wereresuspended in 1 ml lysis buffer and 100 ul aliquots mixed with 100 ulof the luciferase substrate. The reaction mix was left for a 15 minutesadaptation period in the dark before counting for 5 minutes in a TopCount scintillation counter. Luciferase activity is measured as countsper second (CPS) from the scintillation counter. Four independent countswere taken per well.

[0150] Control transfections were set up with no DNA, CaPO₄, an anionicgemini compound (1) and the commercially available lipofection reagentsLipofectAmine™ and Lipotaxi™ at the manufacturers recommendedconcentrations (10, 25, 50, 75, 125 ug/ml and 175, 250, 325, 400 and 500ug/ml respectively).

[0151] The results (FIG. 1) clearly show that the cationic peptide-basedgemini compounds (54), (55) and (56) are very efficient agents forfacilitating the transfection of the luciferase plasmid into HEK293cells at concentrations above 150 ug/ml. In particular compound (54)peaks at 250 ug/ml with a mean count (of 4 independent counts) of over70,000 cps. Compound (55) is most effective at 300 ug/ml with an averagecount of about 45,000 cps. Compound (56) is most effective at 200 ug/mlwith an average count of about 50,000 cps. In contrast the ‘no DNA’negative control gives a background count as do the anionic gemini (1)and the cationic geminis (50 and 52). The CaPO₄ transfection shows avery low count of about 2,000 cps. In comparison FIG. 2 shows theresults for the Lipofectamine transfections which at peak efficiencygave only 12,500 cps (125 ug/ml) and Lipotaxi 2,500 cps (at 175 ug/mland 325 ug/ml).

Example 18

[0152] Transfection of Recombinant Plasmid Expressing Luciferase intoCHO-K1 Cells Using Peptide-Based Gemini Compounds.

[0153] CHO-K1 cells (ATCC: CRL-9618) were seeded into T₂₅-culture flasks(Corning-Costar Buckinghamshire, UK), at 7×105 cells per flask, 24 hoursprior to transfection. The CHO-K1 cells were seeded in 5 ml MEM alphamedium with ribonucleosides and deoxyribonucleosides and supplementedwith 1× L-glutamine and 10% v/v foetal bovine serum (complete medium).The cells were grown at 37° C. in 5% CO₂ in a humidified atmosphere.

[0154] For transfection, 5 ug DNA (luciferase control plasmid) wasincubated with the gemini compounds in water (final volume 400 μl). Thepeptide-based gemini compounds were made up at 1 mg ml⁻¹ in tissueculture grade water and then diluted to the appropriate concentration toa total volume of 200 ul. Following a 30 minute room temperatureincubation, 2.6 ml OPTI-MEM® medium was added and the solution placed onthe cells. Following an overnight incubation at 37° C., the transfectionsolution was replaced with complete medium and the cells incubated at37° C. 24 hours post transfection the cells were detached from the flaskand seeded into 96-well plates at a density of 0.5×10⁵ cells per welland incubated for a further 24 hours at 37° C. Luciferase reporter geneassays were performed according to the manufacturers instructions (RocheDiagnostics, Mannheim, Germany) approximately 48 hours posttransfection. The plates were left for a 15 minutes adaption period inthe dark before counting for 60 seconds in a TopCount NXT counter(Canberra Packard). An average of eight wells were counted pertransfection.

[0155] Control transfections were set up with no DNA, an anionic geminicompound and the commercially available reagent LipofectAmine PLUS™.

[0156] The results, shown in FIG. 3, demonstrate that the cationicpeptide-based compounds 54, 55, and 56 are very efficient agents forfacilitating the transfection of the luciferase plasmid into CHO-K1cells. Using the conditions described above, compound 54 peaks at 30 mMwith a mean count in excess of 1.4×10⁵ counts per second (cps). Compound55 is most effective at 30 mM with an average count of about 2.4×10⁵cps. Compound 56 is most effective at 40 mM with an average count ofabout 1.9×10⁵ cps. In contrast negative controls gave a negligablecount.

Example 19

[0157] Transfection of Recombinant Plasmid Expressing Luciferase intoCHO-K1 Cells Using Peptide-Based Gemini Compounds in Combination withVarious Supplements.

[0158] The transfection ability of the gemini compounds could be furtherenhanced by the addition of a neutral carrier, for example, dioleylphosphatidylethanolamine (DOPE) (Farhood, H., et al (1985) Biochim.Biophys. Acta 1235 289) or a complexing reagent, for example, PLUScompound (Life Technologies Inc.).

[0159]FIG. 4 shows, for example, a 9-fold increase of luciferaseactivity at a 2:1 ratio of compound 55 and DOPE. Transfection mediatedby compound 55 with DOPE in a 2:1 ratio and the addition of 11.6 ul ofPLUS compound lead to a mean count of 6.5×10⁵ cps representing a 12-foldincrease of luciferase activity in comparison to compound 55 alone.Incubation of the PLUS compound with the DNA and combination withcompound 55 alone also lead to a 4-fold increase.

Example 20

[0160] Use of Peptide-Based Gemini Compounds to Facilitate Adhesion ofCells in Culture to the Culture Flask.

[0161] Using normal growth medium and culture conditions (RPMI plus 10%foetal bovine serum; 37° C., 5% CO2 ) but with the addition of 50-60 ugpeptide-based gemini compound per well, it was observed that with thesuspension cell line Jurkat, cells could attach to the bottom surface ofthe plastic culture vessel. In the absence of gemini compounds, theJurkat cells grew in suspension.

1. A peptide-based gemini compound comprising two linked chains:

each chain having: (1) a positively charged hydrophilic head, Q¹ or Q²,formed from one or more amino acids and/or amines (2) a central portion,P¹ or P², having a polypeptide backbone, and (3) a hydrophobic tail, R¹or R², the central sections of each chain being linked together bybridge Y through residues in P¹ and P².
 2. A peptide-based geminicompound according to claim 1 which has the formula (I):

where: A¹ and A⁵ ,which may be the same or different, is a positivelycharged group formed from one or more amino acids or amines joinedtogether in a linear or branched manner; A²/A⁶CH(NH)CO, which may be thesame or different, is derived from an amino acid; p and q, which may bethe same or different, is 0 or 1; X¹/X²CH₂CH(NH)CO, which may be thesame or different, is derived from cysteine (X¹/X²═S), serine orthreonine (X¹/X²═O); A⁴/A⁸CH(NH)CO, which may be the same or different,is derived from serine or threonine; Y is a linker group or a disulphidebond when X¹ and X² is each S; R¹ and R² are C₍₁₀₋₂₀₎ saturated orunsaturated alkyl groups, and W and Z are NH, O, CH₂ or S; or a saltthereof.
 3. A peptide-based gemini compound according to claim 2 whereinthe A¹ and A⁵ groups are bonded by an amide (CONH) bond.
 4. A compoundaccording to claims 2 or 3 wherein A¹/A⁵ are D- or L-amino acidsselected from arginine, lysine, ornithine and histidine.
 5. A compoundaccording to claims 2 to 4 wherein A¹/A⁵ have up to 7 amino acids linkedin a linear or branched chain.
 6. A compound according to claim 5wherein A¹/A⁵ have two or three lysines or ornithines or a combinationof lysine, ornithine, arginine and histidine.
 7. A compound according toany one of claims 2 to 6 wherein the amino acid from which theA²/A⁶CH(NH)CO is derived is serine.
 8. A compound according to any oneof claims 2 to 7 wherein Y is (CH₂)_(m), where m is an integer from 1 to6.
 9. A compound according to any one of claims 2 to 7 wherein Y is adisulphide bond when X¹ and X² is each S.
 10. A compound according toclaim 8 or 9 wherein m is
 2. 11. A compound according to any one ofclaims 2 to 10 wherein R is C₁₂ alkyl.
 12. A compound according to anyone of claims 2 to 11 wherein W and Z are NH.
 13. A compound accordingto any one of claims 2 to 12 wherein the salt is a pharmaceuticallyacceptable salt.
 14. A compound according to any one of claims 1 to 13which is symmetrical, that is A¹ and A⁵ are the same, A² and A⁶ are thesame, A⁴ and A⁸ are the same, R¹ and R² are the same, and W and Z arethe same.
 15. Compound 39: 2-amino-3-{2-[2-amino-2-(1-dodecylcarbamoyl-2-hydroxy-ethylcarbamoyl)-ethylsulphanyl]-ethylsulphonyl}-N-(1-dodecylcarbamoyl-2-hydroxy-ethyl-)-propionamide,and derivatives thereof, compounds 40 to
 58.


16. The compound:


17. The compound:


18. The compound:


19. The compound:


20. The compound:


21. The use of a gemini-based peptide compound as defined in any one ofclaims 1 to 20 in enabling transfection of DNA or RNA or analogs thereofinto a eukaryotic or prokaryotic cell in vivo or in vitro.
 22. The useof a peptide-based gemini compound according to claim 21 wherein thecompound is used in combination with one or more supplements selectedfrom the group consisting of: (i) a neutral carrier; or (ii) acomplexing reagent.
 23. The use according to claim 22 wherein theneutral carrier is dioleyl phosphatidylethanolamine (DOPE).
 24. The useaccording to claim 22 wherein the complexing reagent is PLUS reagent.25. The use according to claim 22 wherein the complexing reagent is apeptide comprising mainly basic amino acids.
 26. The use according toclaim 25 wherein the peptide consists of basic amino acids.
 27. The useaccording to claim 25 or 26 wherein the basic amino acids are selectedfrom lysine and arginine.
 28. The use according to claim 26 wherein thepeptide is polylysine or polyornithine.
 29. A method of transfectingpolynucleotides into cells in vivo for gene therapy, which methodcomprises administering peptide-based gemini compounds of any one ofclaims 1 to 20 together with, or separately from, the gene therapyvector.
 30. The use of a peptide-based gemini compound of any one ofclaims 1 to 20 to facilitate the transfer of a polynucleotide or ananti-infective compounds into prokaryotic or eukaryotic organism for usein anti-infective therapy.
 31. The use of a peptide-based geminicompound of any one of claims 1 to 20 to facilitate the adhesion ofcells in culture to each other or to a solid or semi-solid surface. 32.A process for preparing peptide-based gemini compounds of claim 1 or 2which process comprises adding amino acids or peptides to2-amino-3-{2-[2-amino-2-(1-dodecylcarbamoyl-2-hydroxy-ethylcarbamoyl)-ethylsulphanyl]-ethylsulphonyl}-N-(1-dodecylcarbamoyl-2-hydroxy-ethyl-)-propionamide.